Cystic fibrosis (CF) is a heritable disease that causes altered physiology at mucosal sites; these changes result in chronic infections in the lung, significant gastrointestinal complications as well as dysbiosis of the gut microbiome, although the latter has been less well explored. Here, we describe the longitudinal development of the gut microbiome in a cohort of children with CF (cwCF) from birth through early childhood (0–4 years of age) using 16S rRNA gene amplicon sequencing of stool samples as a surrogate for the gut microbiota. Similar to healthy populations, alpha diversity of the gut microbiome increases significantly with age, but diversity plateaus at ~2 years of age for this CF cohort. Several taxa that have been associated with dysbiosis in CF change with age toward a more healthy-like composition; notable exceptions include Akkermansia , which decreases with age, and Blautia , which increases with age. We also examined the relative abundance and prevalence of nine taxa associated with CF lung disease, several of which persist across early life, highlighting the possibility of the lung being seeded directly from the gut early in life. Finally, we applied the Crohn’s Dysbiosis Index to each sample, and found that high Crohn’s-associated dysbiosis early in life (<2 years) was associated with significantly lower Bacteroides in samples collected from 2 to 4 years of age. Together, these data comprise an observational study that describes the longitudinal development of the CF-associated gut microbiota and suggest that early markers associated with inflammatory bowel disease may shape the later gut microbiota of cwCF. IMPORTANCE Cystic fibrosis is a heritable disease that disrupts ion transport at mucosal surfaces, causing a buildup of mucus and dysregulation of microbial communities in both the lungs and the intestines. Persons with CF are known to have dysbiotic gut microbial communities, but the development of these communities over time beginning at birth has not been thoroughly studied. Here, we describe an observation study following the development of the gut microbiome of cwCF throughout the first 4 years of life, during the critical window of both gut microbiome and immune development. Our findings indicate the possibility of the gut microbiota as a reservoir of airway pathogens and a surprisingly early indication of a microbiota associated with inflammatory bowel disease.

The healthy human infant gut microbiome undergoes stereotypical changes in taxonomic composition between birth and maturation to an adult-like stable state. During this time, extensive communication between microbiota and the host immune system contributes to health status later in life. Although there are many reported associations between microbiota compositional alterations and disease in adults, less is known about how microbiome development is altered in pediatric diseases. One pediatric disease linked to altered gut microbiota composition is cystic fibrosis (CF), a multi-organ genetic disease involving impaired chloride secretion across epithelia and heightened inflammation both in the gut and at other body sites. Here, we use shotgun metagenomics to profile the strain-level composition and developmental dynamics of the infant fecal microbiota from several CF and non-CF longitudinal cohorts spanning from birth to greater than 36 months of life. We identify a set of keystone species whose prevalence and abundance reproducibly define microbiota development in early life in non-CF infants, but are missing or decreased in relative abundance in infants with CF. The consequences of these CF-specific differences in gut microbiota composition and dynamics are a delayed pattern of microbiota maturation, persistent entrenchment in a transitional developmental phase, and subsequent failure to attain an adult-like stable microbiota. We also detect the increased relative abundance of oral-derived bacteria and higher levels of fungi in CF, features that are associated with decreased gut bacterial density in inflammatory bowel diseases. Our results define key differences in the gut microbiota during ontogeny in CF and suggest the potential for directed therapies to overcome developmental delays in microbiota maturation.

Persons with cystic fibrosis, starting in early life, have intestinal microbiome dysbiosis characterized in part by a decreased relative abundance of the genus Bacteroides. Bacteroides is a major producer of the intestinal short chain fatty acid (SCFA) propionate. We demonstrate here that CFTR-/- Caco-2 intestinal epithelial cells are responsive to the anti-inflammatory effects of propionate. Furthermore, Bacteroides isolates inhibit the IL-1β-induced inflammatory response of CFTR-/- Caco-2 intestinal epithelial cells and do so in a propionate-dependent manner. Bacteroides isolates also produce low levels of butyrate; this SCFA is positively correlated with inhibition of the inflammatory response. Finally, the introduction of Bacteroides-supplemented stool from infants with CF into the gut of CftrF508del mice results in an increase in propionate in the stool as well as the reduction in several systemic pro-inflammatory cytokines. Bacteroides supplementation also reduced the fecal relative abundance of E. coli, indicating a potential interaction between these two microbes, consistent with previous clinical studies. Together, our data indicate the important role of Bacteroides and Bacteroides-derived propionate in the context of the developing microbiome in infants and children with CF, which could help explain the observed gut-lung axis in CF.

Cystic Fibrosis is a heritable disease that causes altered physiology at mucosal sites; these changes result in chronic infection in the lung, significant gastrointestinal complications as well as dysbiosis of the gut microbiome, although the latter has been less well explored. Here, we describe the longitudinal development of the gut microbiome in a cohort of children with cystic fibrosis (cwCF) from birth through early childhood (0-4 years of age) using 16S rRNA gene amplicon sequencing of stool samples as a surrogate for the gut microbiota. Similar to healthy populations, alpha diversity of the gut microbiome increases significantly with age, but diversity plateaus ~2 years of age for this CF cohort. Several taxa that have been associated with dysbiosis in CF change with age towards a more healthy-like composition; notable exceptions include Akkermansia, which decreases with age, and Blautia, which increases with age. We also examined the relative abundance and prevalence of nine taxa associated with CF lung disease, several of which persist across early life, highlighting the possibility of the lung being seeded directly from the gut early in life. Finally, we applied the Crohn's dysbiosis index to each sample, and found that high Crohn's-associated dysbiosis early in life (<2 years) was associated with significantly lower Bacteroides in samples collected from 2-4 years of age. Together, these data indicate a persisting dysbiosis in the gut microbiota as well as markers associated with inflammatory bowel disease early in life for cwCF.

The gut physiology of pediatric and adult persons with cystic fibrosis (pwCF) is altered relative to healthy persons. The CF gut is characterized, in part, as having excess mucus, increased fat content, acidic pH, increased inflammation, increased antibiotic perturbation and the potential for increased oxygen availability. These physiological differences shift nutritional availability and the local environment for intestinal microbes, thus likely driving significant changes in microbial metabolism, colonization and competition with other microbes. The impact of any specific change in this physiological landscape is difficult to parse using human or animal studies. Thus, we have developed a novel culture medium representative of the CF gut environment, inclusive of all the aforementioned features. This medium, called CF-MiPro, maintains CF gut microbiome communities, while significantly shifting non-CF gut microbiome communities toward a CF-like microbial profile, characterized by low Bacteroidetes and high Proteobacteria abundance. This medium is able to maintain this culture composition for up to 5 days of passage. Additionally, microbial communities passaged in CF-MiPro produce significantly less immunomodulatory short chain fatty acids (SCFA), including propionate and butyrate, than communities passaged in MiPro, a culture medium representative of healthy gut physiology, confirming not only a shift in microbial composition but altered community function. Our results support the potential for this in vitro culture medium as a new tool for the study of gut dysbiosis in CF.ImportanceCystic fibrosis is an autosomal recessive disease that disrupts ion transport at mucosal surfaces, leading to mucus accumulation and altered physiology of both the lungs and the intestines, among other organs, with the resulting altered environment contributing to an imbalance of microbial communities. Culture media representative of the CF airway have been developed and validated; however, no such medium exists for modeling the CF intestine. Here, we develop and validate a first-generation culture medium inclusive of features that are altered in the CF colon. Our findings suggest this novel medium, called CF-MiPro, as a maintenance medium for CF gut microbiome samples and a flexible tool for studying key drivers of CF-associated gut dysbiosis.